PROJECT SUMMARY It has been shown that in utero tobacco exposure increases offspring risk for a number of diseases including obesity and diabetes, which are significant burdens on the healthcare system in the US and worldwide. As part of a pilot study in Kentucky, 30% of pregnant mothers reported that they actively smoked cigarettes during their pregnancy, and it was shown that tissues from babies born to smoking mothers had altered metabolic gene expression and DNA methylation levels compared to babies born to nonsmokers. Further, primary fibroblasts isolated from babies born to smokers took up more lipids and had impaired insulin signaling compared to cells isolated from babies born to nonsmokers. These observations form the basis of the hypotheses that 1) the DNA methylation pattern and expression of key energy metabolism genes and their resulting proteins will be significantly altered in the neonatal tissue of babies born to active smokers compared to those born to nonsmokers, 2) living cells isolated from babies born to active smokers will be functionally dysregulated compared to cells taken from babies of nonsmokers, and 3) biomarkers that are identified will be predictive of increased growth rates and pediatric obesity. The goal of Specific Aim 1 is to establish biomarkers and potential mechanisms contributing to offspring obesity and diabetes risk resulting from in utero cigarette exposure. Gene expression and epigenetic alterations will be quantified in neonatal tissue in order to establish differences between exposure groups. Specific Aim 2 will determine the extent to which neonatal cells are uniquely programmed by in utero smoke exposure. Primary skin fibroblasts will be isolated from neonates and grown in culture. Glucose uptake and susceptibility to differentiate into adipocyte-like cells will be assessed as it is likely these cells are already dysfunctional at birth. Finally, the goal of Specific Aim 3 is to use gene expression data, DNA methylation changes, and cell function measures as potential biomarkers that will predict increased growth rates and obesity susceptibility in male infants that were exposed to tobacco smoke in utero. Using primary fibroblasts isolated from neonates in order to measure adipogenesis and glucose uptake in live cells is not currently possible using any other model system to explore the effects of maternal smoking on offspring. The proposed study provides a human system to demonstrate the mechanisms through which tobacco smoke exposure in utero contributes to the epidemic of childhood obesity. Research findings and this system could also provide an innovative approach to understand the effects of various other in utero exposures on metabolic outcomes in children. Future work can also utilize this model to investigate new preventative and intervention approaches targeting these pathways.